Evaluation of Vascular Smooth Muscle Cell Lineages Derived from the Transgenic Mice Expressing Temperature-Sensitive Simian Virus 40 T Antigen Targeted to Smooth Muscle

ABSTRACT

BACKGROUND AND OBJECTIVES: As a part of efforts to evaluate effectiveness of aorta cell lineages derived from the transgenic mice in which expression of the temperature-sensitive SV40 large T antigen has been targeted to vascular smooth muscle, localization of the T antigen gene in chromosome of the established cell lineages was characterized. Expression pattern of p53 in an aorta cell line or those of r-actin and the T antigen in the various tissues of the transgenic mice, respectively, were also examined. MATERIALS AND METHODS: Chromosomal DNA obtained from the aorta cell lines, which were derived from the transgenic mice, was analyzed by genomic Southern blot method to identify the transgene in genome. mRNA was prepared from the various tissues of the transgenic mouse and was examined by Northern blot analysis to detect expression of r-actin gene. Reverse transcription polymerase chain reaction (RT-PCR) was also performed to examine transcription pattern of p53 gene in an aorta cell line. Immunohistology for detecting the T antigen expression in the tissues of the transgenic mouse was also carried out. RESULTS: Correct integration of the SV40 T antigen transgene in chromosome was observed in two aorta cell lines, although their copy numbers inserted were different from each other. Alternative splicing of the primary p53 RNA was identified in the aorta cells when cultured at the restrictive temperature, but not in the cells at permissive temperature. Several tissues of the transgenic mouse showed expression of the viral oncoprotein T antigen. CONCLUSION: An established aorta cell line may be useful model to study the mechanism regulating the proliferation of vascular smooth cells in mice. Mutant form (s) of the p53 tumor suppressor protein generated by the translation of the alternatively spliced mRNA might be involved in the blockade of apoptosis in some aorta cells when cultured at the restrictive temperature. However, expression of the viral gene in the tissues of the transgenic mice seemed not to be precisely regulated by temperature-dependent manner.